Expression of Heterologous Proteins in Plant Seeds
The expression of heterologous proteins in plant seeds offers the possibility of, for example, producing large quantities of easily harvested polypeptides, and of expressing proteins that improve their grain quality. Discussions of this concept can be found in U.S. Pat. No. 5,714,474 (xe2x80x9cProduction of enzymes in seeds and their usesxe2x80x9d).
Hordein Storage Proteins
Barley seed storage proteins account for about 8 to 15% of the dry weight of the mature barley grain. The major seed storage proteins in barley are alcohol-soluble prolamines, termed hordeins, which are classified into two major groups, B and C, and two minor groups, D and xcex3 (Shewry 1993). Depending on nitrogen levels, these four groups account for about 35 to 55% of total barley seed protein. The B-and C-hordeins account for about 70 to 80% and 10 to 20%, respectively, of the total hordein fraction, with small amounts of D- (2-4%) and xcex3-hordeins (not precisely determined). The B-, D- and xcex3-hordeins are sulfur-rich prolamines while the C hordeins are sulfur-poor prolamines (Bright and Shewry 1983). The hordeins are coordinately synthesized in the developing starchy endosperm tissue (Giese et al. 1983; Sxc3x8rensen et al. 1989). They are cotranslationally transported into the lumen of the rough endoplamic reticulum, with simultaneous cleavage of the signal peptide, and are ultimately deposited into protein bodies (Cameron-Mills 1980; Cameron-Mills and von Wettstein 1980; Cameron-Mills and Madrid 1989).
Genetic analyses show that all hordeins are encoded by structural genes on chromosome 5 (1H) of barley; the Hor1, Hor2, Hor3 and Hor5 loci on chromosome 5 encode the C-, B-, D-, and xcex3-hordein polypeptides, respectively (Jensen et al., 1980; Shewry et al. 1980; Blake et al. 1982; Shewry et al. 1983; Shewry and Parmar 1987). The genes for B-, C- and D-hordeins have been isolated and characterized (Brandt et al. 1985; Forde et al. 1985; Rasmussen and Brandt 1986; Sxc3x8rensen et al. 1996). The B- and C-hordeins are encoded by multigene families comprising 10 to 20 members while D-hordein is encoded by a single gene (Brandt et al. 1985; Rasmussen and Brandt 1986; Sxc3x8rensen et al. 1996). The regulation and expression of these hordein promoters have been studied by transient expression assays (Entwistle et al. 1991; Mxc3xcller and Knudsen 1993; Sxc3x8rensen et al. 1996) in barley endosperm. As determined by these assays using promoter-uidA fusions, the D-hordein promoter is 3- to 5-fold more active than the B- or C-hordein promoters tested (Sxc3x8rensen et al. 1996). The B-hordein promoter has also been studied using stable tobacco transformation with promoter-cat fusions (Marris et al. 1988).
Although the genes for B-, C- and D-hordeins have been isolated and characterized, their regulation and expression have been studied only in transient expression assays in barley and in stably transformed tobacco (Brandt et al., 1985; Forde et al., 1985; Marris et al., 1988 Sxc3x8rensen et al., 1996).
In barley, wheat and maize, the major highly insoluble prolamin storage proteins are synthesized on polysomes closely associated with the endoplasmic reticulum (ER). (See Seeds: Physiology of Development and Germination, 2nd ed., eds. Bewley and Black, Plenum Press, New York, 1994). Newly synthesized proteins pass through the ER membrane into the lumen, where they aggregate into small particles, which eventually form larger aggregates and protein bodies (which can be observed in electron micrographs).
In wheat, two different types of protein bodies accumulate independently within the developing endosperm: low density bodies that develop earlier and high density bodies that develop later and are derived from the ER. The high density proteins are formed when aggregation of proteins inside the lumen of the ER puts a strain on the membrane and cause it to rupture. The membrane may reform free of the protein aggregate, after an interval in which the protein body itself is not bounded by a membrane. In other cereals besides wheat and barley, such as millet, rice, maize and sorghum, the protein bodies remain as distinct membrane-bound entities even in mature seed.
The present invention provides recombinant nucleic acid molecules that employ a seed maturation specific promoter linked to a polypeptide, and particularly to a construct that also includes a signal sequence that targets a linked polypeptide to an intracellular body, such as a protein body. Such constructs may be represented as P-X or P-SS-X wherein P is a seed maturation-specific promoter, SS is a signal sequence, such as a sequence that targets a linked polypeptide to an intracellular body, and X is a polypeptide to be expressed in seed or a plant embryo. In particular embodiments, X is a non-storage protein, which is targeted to a protein storage body. Seeds expressing polypeptides of interest may be harvested at preselected times that have been found to provide greatest expression or stability of the polypeptide.
In particular embodiments, the invention provides recombinant nucleic acid molecules that employ hordein promoters, such as hordein B1 and D promoters, to direct expression of polypeptides in seeds of transgenic plants, including monocot plants. In particular, the invention provides nucleic acid molecules in which a hordein signal sequence is operably linked to a nucleic acid sequence encoding the polypeptide of interest. It is shown that inclusion of a hordein signal sequence can significantly increase the levels of the expressed polypeptide in the plant seed. A wide range of polypeptides may be expressed in plant seeds in this manner, including pharmaceuticals such as insulin, interferons, erythropoietin and interleukins, and nutritional supplements.
The nucleic acid molecules provided by this aspect of the invention may be represented as
Ph-hSS-X
wherein Ph is a hordein promoter, hSS is a hordein signal sequence, and X is a nucleic acid molecule encoding a polypeptide (particularly a polypeptide that is not a seed storage protein), and where Ph, hSS and X are operably linked. Nucleic acid molecules lacking the hordein signal sequence may be represented as:
Ph-X.
The invention provides transgenic plants comprising these nucleic acid molecules, as well as seeds of these transgenic plants, which are useful as a source of the expressed polypeptide, or may improve the quality of the grain.
In particular embodiments of the invention, the transgenic plants provided are stably transformed monocotyledenous plant, for example cereal plants, such as barley or wheat. In particular embodiments, the invention provides stably transformed barley plants from genotypes including: Harrington, Morex, Crystal, Stander, Moravian III, Galena, Salome, Steptoe, Klages and Baronesse. The invention also provides stably transformed wheat plants from genotypes including: Anza, Karl, Bobwhite and Yecora Rojo. Most of these genotypes are not amenable to conventional transformation procedures. Accordingly, in order to enable production of stably transformed plants belonging to these genotypes, the invention also provides a transformation method that, in conjunction with the nucleic acid molecules provided, may be employed to produce the stably transformed plants. This transformation method is based upon the production of green regenerable tissues from zygotic plant embryos, and may be used to transform any monocot species, including maize, wheat, barley, rice, oat, rye, millet, sorghum, tricalate, turfgrass and forage grasses.
The transformation method comprises
(a) placing an immature zygotic embryo of the selected monocot plant on plant growth medium comprising maltose as a sugar source, an auxin at a concentration of about 0.1 mg/L to about 5 mg/L, a cytokinin at a concentration of 0 mg/L to about 5 mg/L and copper at a concentration of about 0.1 xcexcM to about 50 xcexcM, and incubating in dim light conditions so as to form green regenerative tissue;
(b) introducing a nucleic acid molecule into the tissue by to produce transformed tissue; the nucleic acid may be either
P-hSS-X,
P-X,
Ph-hSS-X, or
Ph-X
xe2x80x83wherein P is a seed maturation-specific promoter, SS is a signal sequence that tragets a polypeptide to an intracellular body (such as a protein body or vacuole), Ph is a hordein promoter (a particular seed maturation specific promoter), hSS is a hordein signal sequence, and X is a nucleic acid molecule encoding the selected polypeptide (which may be other than a seed storage protein), and where Ph, hSS and X (or Ph and X) are operably linked;
(c) incubating the transformed tissue on the plant growth medium such that green structures are observed on the transformed material;
(d) regenerating at least one transformed plant from the green structures; and
(f) growing the transformed plant to produce seed.
The invention also provides seeds of stably transformed plants that express the selected polypeptide in their seed. Another aspect of the invention is a method of expressing a polypeptide in seed of a monocotyledenous plant, comprising providing a monocotyledenous plant that is stably transformed with a nucleic acid molecule of structure Ph-hSS-X, or Ph-X, and growing the plants under conditions effective to produce seed and express the polypeptide in the seed. The polypeptide can be used to improve the quality of the grain, or it can be extracted from the seed at a time of maximum expression or stability to be used for other purposes.